Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca 2+ dependent manner. Cytosolic Ca 2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca 2+ increase, thereby suppressing induction of autophagy.

Sepsis caused by Streptococcus pyogenes is a life-threatening condition. Blood vessel endothelial cells should serve as a barrier to infection, although we recently reported that endothelial cells allow intracellular GAS proliferation due to defective xenophagy.

Tartary buckwheat is used as an ingredient in flour and tea, as well as in traditional Chinese medicine for its antioxidant effects. Here, we found that an ethanol extract of tartary buckwheat (TBE) potently induced autophagy flux in HeLa cells by suppressing mTORC1 activity, as revealed by dephosphorylation of the mTORC1 substrates Ulk1, S6K, and 4EBP, as well as by the nuclear translocation of transcriptional factor EB. In addition to non-selective bulk autophagy, TBE also induced aggrephagy, which is defined as autophagy against aggregated proteins. Quercetin is a flavonol found at high levels in TBE. We showed that quercetin induced both non-selective bulk autophagy and aggrephagy. These effects were also observed in Huh-7 cells derived from hepatocytes. Thus, aggrephagy induction by TBE and quercetin may relieve alcoholic hepatitis, which is closely linked to the accumulation of protein aggregations called Mallory–Denk bodies.

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Streptococcus pyogenes, also known as group A streptococcus (GAS), is one of most common human pathogens. Severe life-threatening disease, such as sepsis, results from the breakdown of blood vessel barrier. Although GAS has been identified as an extracellular bacterium, internalization of GAS by many eukaryotic cells may provide a mechanism of clinical severe damage due to evading from immune surveillance or antibiotic treatment. Previous report showed that GAS can be efficiently killed by autophagy (an intracellular innate immune system) in some nonphagocytes including epithelial cells. In this study, we showed that invasive GAS, but not noninvasive GAS strains, multiplied inside endothelial cells and bacterial growth reached plateau at 6 ~ 8 h. The intracellular sites of multiplication of GAS were first in vesicles and then in cytoplasm where most of the GAS was liberated from vesicle by streptolysin before endosomal degradation. GAS further induced autophagy formation but only a small portion of GAS was completely enveloped by GAS-containing autophagosome-like vacuoles at 6 h. Furthermore, endothelial cells underwent a caspase-indepandent cell death caused by persistent multiplication of GAS. The interrelationship between autophagy induction, cell death, and GAS multiplication remains further investigation. Our studies demonstrate that the insufficient autophagy induction during invasive GAS infection, particularly in endothelial cells, results in multiplication of GAS.

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